Abstract

We demonstrate the impact of a disrupted molecular clock in Bmal1-deficient (Bmal1−/−) mice on migration of neural progenitor cells (NPCs). Proliferation of NPCs in rostral migratory stream (RMS) was reduced in Bmal1−/− mice, consistent with our earlier studies on adult neurogenesis in hippocampus. However, a significantly higher number of NPCs from Bmal1−/− mice reached the olfactory bulb as compared to wild-type littermates (Bmal1+/+ mice), indicating a higher migration velocity in Bmal1−/− mice. In isolated NPCs from Bmal1−/− mice, not only migration velocity and expression pattern of genes involved in detoxification of reactive oxygen species were affected, but also RNA oxidation of catalase was increased and catalase protein levels were decreased. Bmal1+/+ migration phenotype could be restored by treatment with catalase, while treatment of NPCs from Bmal1+/+ mice with hydrogen peroxide mimicked Bmal1−/− migration phenotype. Thus, we conclude that Bmal1 deficiency affects NPC migration as a consequence of dysregulated detoxification of reactive oxygen species.

Figure S5. Treatment of NPCs from Bmal1-/- mice with hydrogen peroxide and NPCs from Bmal1+/+ mice with catalase does not affect migration. a) Representative photomicrographs of NPCs from Bmal1-/- mice (-/-) treated with vehicle (control) or 80 µM hydrogen peroxide (H2O2) for 24 h. Scale bar: 200 µm. (b) Time course of migration distance and velocity is not different between vehicle (control) and treatment with 80 µM H2O2 during the first 24 h after seeding. n=3 mice per group. (c) Representative photomicrographs of NPCs from Bmal1++ mice (+/+) treated with vehicle (control) or 500 U/ml catalase (catalase) for 24 h. Scale bar: 200 µm. (d) Time course of migration distance and velocity is not different between vehicle (control) and treatment with 500 U/ml catalase during the first 24 h after seeding. n=3 mice per group (TIF 30992 KB)

Figure S6 Treatment of NPCs derived from Bmal1-/- mice with N-acetylcysteine does not affect migration. Neurospheres derived from Bmal1-/- mice were seeded in migration medium supplemented with different concentrations of N-acetylcysteine or vehicle (control) and continuously recorded during the first 24 h (TIF 25289 KB)

Appendix Video 1 Migration of NPCs derived from Bmal1
+/+ mice. Neurospheres were recorded continuously during the first 24 hours after culture in migration medium. Time interval between phase-contrast images at 100x magnification was 60 min (AVI 38712 KB)

Appendix Video 2 Migration of NPCs derived from Bmal1
-/- mice. Neurospheres were recorded continuously during the first 24 hours after seeding in migration medium. Time interval between phase-contrast images at 100x magnification was 60 min (AVI 38574 KB)

Appendix Video 3 Migration of NPCs derived from Bmal1
+/+ mice in the presence of hydrogen peroxide. Neurospheres were recorded continuously during the first 24 hours after seeding in migration medium in the presence of 80 µM hydrogen peroxide (H
2O
2). Time interval between phase contrast images at 100x magnification was 60 min (AVI 38634 KB)

Appendix Video 4 Migration of NPCs derived from Bmal1
-/- mice in the presence of catalase. Neurospheres were recorded continuously during the first 24 hours after seeding in migration medium in the presence of 500 U/ml catalase. Time interval between phase contrast images at 100x magnification was 60 min (AVI 38072 KB)

Guruprasad K, Reddy BV, Pandit MW (1990) Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Eng 4(2):155–161
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